A number of DNA viruses have been genetically engineered to direct the expression of heterologous proteins in host cell systems (e.g., vaccinia virus, baculovirus, etc.). Recently, similar advances have been made with positive-strand RNA viruses (e.g., poliovirus). The expression products of these constructs, i.e., the heterologous gene product or the chimeric virus which expresses the heterologous gene product, are thought to be potentially useful in vaccine formulations (either subunit or whole virus vaccines). One drawback to the use of viruses such as vaccinia for constructing recombinant or chimeric viruses for use in vaccines is the lack of variation in its major epitopes. This lack of variability in the viral strains places strict limitations on the repeated use of chimeric vaccinia, in that multiple vaccinations will generate host-resistance to the strain so that the inoculated virus cannot infect the host. Inoculation of a resistant individual with chimeric vaccinia will, therefore, not induce immune stimulation.
By contrast, the negative-strand RNA viruses, are attractive candidates for constructing chimeric viruses for use in vaccines. Negative-strand RNA viruses, for example, influenza, are desirable because their wide genetic variability allows for the construction of a vast repertoire of vaccine formulations which stimulate immunity without risk of developing a tolerance.
2.1 Negative-Strand RNA Viruses
The virus families containing enveloped single-stranded RNA of the negative-sense genome are classified into groups having non-segmented genomes (Paramyxoviridae, Rhabdoviridae) or those having segmented genomes (Orthomyxoviridae, Bunyaviridae and Arenaviridae). The Paramyxoviridae and Orthomyxoviridae families are described in detail below and used in the examples herein. The Paramyxoviridae family contains the viruses of Newcastle disease Virus (NDV), parainfluenza virus, Sendai virus, simian virus 5, and mumps virus. The Orthomyxoviridae family contains the viruses of influenza, types A, B and C viruses, as well as Thogoto and Dhori viruses and infectious salmon anemia virus.
2.1.1 Influenza Virus
The influenza virions comprise an internal ribonucleoprotein core (a helical nucleocapsid) containing the single-stranded RNA genome, and an outer lipoprotein envelope lined inside by a matrix protein (M1). The segmented genome of influenza A virus consists of eight molecules (seven for influenza C) of linear, negative polarity, single-stranded RNAs which encode ten polypeptides, including: the RNA-dependent RNA polymerase proteins (PB2, PB1 and PA) and nucleoprotein (NP) which form the nucleocapsid; the matrix membrane proteins (M1, M2); two surface glycoproteins which project from the lipid containing envelope: hemagglutinin (HA) and neuraminidase (NA); the nonstructural protein (NS1) and nuclear export protein (NEP). Transcription and replication of the genome takes place in the nucleus and assembly occurs via budding on the plasma membrane. The viruses can reassort genes during mixed infections.
Influenza virus adsorbs to cells via HA binding activity to sialyloligosaccharides in cell membrane glycoproteins and glycolipids. Following endocytosis of the virion, a conformational change in the HA molecule occurs within the cellular endosome which facilitates membrane fusion, thus triggering uncoating. The nucleocapsid migrates to the nucleus where viral mRNA is transcribed. Viral mRNA is transcribed by a unique mechanism in which viral endonuclease cleaves the capped 5′-terminus from cellular heterologous mRNAs which then serve as primers for transcription of viral RNA templates by the viral transcriptase. Transcripts terminate at sites 15 to 22 bases from the ends of their templates, where oligo(U) sequences act as signals for the addition of poly(A) tracts. The viral RNA transcripts then migrate to the cell membrane and associate with the newly transcribed, transmembrane viral proteins. NA then cleaves sialy residues from the carbohydrate moieties of membrane bound glycoproteins resulting in encapsulation and cellular release of the progeny virus. Of the eight viral RNA molecules so produced, six are monocistronic messages that are translated directly into the proteins representing HA, NA, NP and the viral polymerase proteins, PB2, PB1 and PA. The other two transcripts undergo splicing, each yielding two mRNAs which are translated in different reading frames to produce M1, M2, NS1 and NEP. In other words, the eight viral RNA segments code for ten proteins: nine structural and one nonstructural. A summary of the genes of the influenza virus and their protein products is shown in Table 1 below.
TABLE 1INFLUENZA VIRUS GENOME RNA SEGMENTS AND CODING ASSIGNMENTSaLengthbEncodedLengthdMoleculesSegment(Nucleotides)Polypeptidec(Amino Acids)Per VirionComments12341PB275930-60RNA transcriptase component;host cell RNA cap binding22341PB175730-60RNA transcriptase component;initiation of transcription32233PA71630-60RNA transcriptase component41778HA566500Hemagglutinin; trimer; envelopeglycoprotein; mediatesattachment to cells51565NP4981000Nucleoprotein; associated withRNA; structural component ofRNA transcriptase61413NA454100Neuraminidase; tetramer;envelope glycoprotein71027M12523000Matrix protein; lines inside ofenvelopeM296?Structural protein in plasmamembrane; spliced mRNA8890NS1230Nonstructural protein;NEP121?Nuclear export protein; splicedmRNAaAdapted from R. A. Lamb and P. W. Choppin (1983), Annual Review of Biochemistry, Volume 52, 467-506.bFor A/PR/8/34 straincDetermined by biochemical and genetic approachesdDetermined by nucleotide sequence analysis and protein sequencing
The pathogenicity of influenza viruses is modulated by multiple virus and host factors. Among the host factors that fight virus infections, the type I interferon (IFNα/β) system represents a powerful antiviral innate defense mechanism which was established relatively early in the evolution of eukaryotic organisms (Garcia-Sastre, 2002, Microbes Infect 4:647-55). The antiviral IFNα/β system involves three major steps: (i) detection of viral infection and IFNα/β secretion, (ii) binding of IFNα/β to its receptors and transcriptional induction of IFNα/β-stimulated genes, and (iii) synthesis of antiviral enzymes and proteins. Most viruses, however, have acquired specific genetic information encoding IFNα/β antagonist molecules, which effectively block one or more steps of the antiviral IFNα/β system. Influenza A viruses express a non-structural protein in infected cells, the NS1 protein (described in detail, infra), which counteracts the cellular IFNα/β response (Garcia-Sastre et al., 1998, Virology 252:324-30).
2.1.1.1 High-Pathogenenicity Avian Influenza
In recent years, outbreaks of high pathogenic avian influenza (HPAI) have been reported in Asia and Europe (Kawaoka et al., 2005, Natl. Rev. Microbiol. 3:591-600; Koopmans et al., 2004, Lancet 363:587-593). Outbreaks involving influenza A, subtype H5N1 or H7N7 viruses resulted in lethal infections in domestic poultry, and the death of a limited number of human cases (Tweed et al., 2004, Emerg. Infec. Dis. 10:2196-2199). The current H5N1 viruses have been circulating among poultry within China in recent years (Chen et al., 2005, Nature 436:191-192), and while migratory birds are considered to be the primary reservoir of these viruses, transmission from infected poultry back to migratory birds is believed to have contributed to their increased geographical distribution. Currently, the H5N1 virus has emerged from Asia, spreading across Europe and Africa (Enserink, 2006, Science, 311:932). Wholesale culling of poultry has been shown to be a successful strategy in eradicating H5N1 outbreaks in Hong Kong in 1997 and the Netherlands in 2003 (Lipatov et al., 2004, J. Virol. 78:8951-8959). As human victims of recent HPAI outbreaks have had close contact with infected poultry, it follows that the prevention of interspecies transmission of avian influenza viruses (AIV) may be accomplished by the eradication of AIV in poultry through slaughter. However, for economic and practical reasons, the destruction of infected poultry alone is no longer considered the method of choice in the control of this disease. In addition, for ethical and ecological reasons, the culling of migratory wildfowl is considered an unacceptable practice. Recently, OIE (World Organization for Animal Health) and FAO (Food and Agriculture Organization of the United Nations) recommended that vaccination of poultry should be considered for the control of AIV. In addition, it has been reported that vaccination of chickens with inactivated H5 vaccine was successful in the interruption of virus transmission in a field study (Ellis et al., 2004, Avian Pathol. 33:405-412). Recently, China has accepted vaccination as a component of their AIV control program.
The possibility of that the highly pathogenic H5N1 strain can become transmissible between humans is referenced in terms of a global pandemic, with the WHO unwilling to estimate the global mortality should the H5N1 virus recombine to human form. Therefore, the need for a method of management of H5N1 infection in agricultural stocks, from which most transmissions to humans are believed to have arisen, is clear.
2.1.2 Newcastle Disease Virus
The Newcastle Disease Virus is an enveloped virus containing a linear, single-strand, nonsegmented, negative sense RNA genome. The genomic RNA contains genes in the order of 3′-N-P-M-F-HN-L, described in further detail below. The genomic RNA also contains a leader sequence at the 3′ end.
The structural elements of the virion include the virus envelope which is a lipid bilayer derived from the cell plasma membrane. The glycoprotein, hemagglutinin-neuraminidase (HN) protrudes from the envelope providing both hemagglutinin (e.g., receptor binding/fusogenic) and neuraminidase activities. The fusion glycoprotein (F), which also interacts with the viral membrane, is first produced as an inactive precursor, then cleaved post-translationally to produce two disulfide linked polypeptides. The active F protein is involved in penetration of NDV into host cells by facilitating fusion of the viral envelope with the host cell plasma membrane. The matrix protein (M), is involved with viral assembly, and interacts with both the viral membrane as well as the nucleocapsid proteins.
The main protein subunit of the nucleocapsid is the nucleocapsid protein (N) which confers helical symmetry on the capsid. In association with the nucleocapsid are the P and L proteins. The phosphoprotein (P), which is subject to phosphorylation, is thought to play a regulatory role in transcription, and may also be involved in methylation, phosphorylation and polyadenylation. The L gene, which encodes an RNA-dependent RNA polymerase, is required for viral RNA synthesis together with the P protein. The L protein, which takes up nearly half of the coding capacity of the viral genome is the largest of the viral proteins, and plays an important role in both transcription and replication.
The replication of all negative-strand RNA viruses, including NDV, is complicated by the absence of cellular machinery required to replicate RNA. Additionally, the negative-strand genome can not be translated directly into protein, but must first be transcribed into a positive-strand (mRNA) copy. Therefore, upon entry into a host cell, the virus can not synthesize the required RNA-dependent RNA polymerase. The L, P and N proteins must enter the cell along with the genome on infection.
It is hypothesized that most or all of the viral proteins that transcribe NDV mRNA also carry out their replication. The mechanism that regulates the alternative uses (i.e., transcription or replication) of the same complement of proteins has not been clearly identified but appears to involve the abundance of free forms of one or more of the nucleocapsid proteins, in particular, the N. Directly following penetration of the virus, transcription is initiated by the L protein using the negative-sense RNA in the nucleocapsid as a template. Viral RNA synthesis is regulated such that it produces monocistronic mRNAs during transcription.
Following transcription, virus genome replication is the second essential event in infection by negative-strand RNA viruses. As with other negative-strand RNA viruses, virus genome replication in Newcastle disease virus (NDV) is mediated by virus-specified proteins. The first products of replicative RNA synthesis are complementary copies (i.e., plus-polarity) of NDV genome RNA (cRNA). These plus-stranded copies (anti-genomes) differ from the plus-strand mRNA transcripts in the structure of their termini. Unlike the mRNA transcripts, the anti-genomic cRNAs are not capped and methylated at the 5′ termini, and are not truncated and polyadenylated at the 3′ termini. The cRNAs are coterminal with their negative strand templates and contain all the genetic information in each genomic RNA segment in the complementary form. The cRNAs serve as templates for the synthesis of NDV negative-strand viral genomes (vRNAs).
Both the NDV negative strand genomes (vRNAs) and antigenomes (cRNAs) are encapsidated by nucleocapsid proteins; the only unencapsidated RNA species are virus mRNAs. For NDV, the cytoplasm is the site of virus RNA replication, just as it is the site for transcription. Assembly of the viral components appears to take place at the host cell plasma membrane and mature virus is released by budding.
2.2 Immunogenic Formulations
Recombinant DNA technology and “reverse genetics” engineering techniques afford a unique approach to the production of recombinant viruses for the use in immunogenic formulations. In particular, the present invention provides for a method to engineer a negative-strand RNA virus such that it expresses, or displays, not only native viral antigens, but also any antigen that may be designed to incorporate into the viral protein coat. Of particular interest are antigens derived from infectious organisms other than influenza. In this manner a single virus may be engineered as an immunogenic compound useful to illicit, activate or induce an immune response which would afford protection against at least two pathogens. Such a chimeric virus may be further engineered when necessary to modify their virulence, i.e., so that they may be attenuated or further attenuated. Attenuated influenza viruses are beneficial because they are immunogenic and capable of replication, but not pathogenic.
Live vaccines are thought to induce improved cross-reactive cell-mediated cytotoxicity as well as a humoral antibody response, providing better protection than inactivated vaccines (Gorse and Belshe, 1990, J. Clin. Microbiol. 28:2539-2550; and Gorse et al., 1995, J. Infect. Dis. 172:1-10). Secondly, protective immunity to viral diseases is likely to involve mucosal IgA response which is not seen with traditional intramuscularly administered vaccines (Nelson et al., 1998, Vaccine 16:1306-1313). Finally, live vaccines also have the advantage of intranasal administration which avoids the swelling and muscle soreness occasionally associated with the intramuscular administration of inactivated adjuvanted vaccines. These live vaccines have been reported to induce not only humoral responses against homotypic influenza virus but also crossreactive cell-mediated cytotoxicity. Thus, the invention offers the potential for the development of new and more effective immune formulations, e.g., vaccine formulations, for the diagnosis, prevention, management or treatment of both viral and non-viral pathogens.